This application is directed to nucleosides, oligonucleotides and oligonucleosides that are functionalized with carbamate moieties. The carbamate moieties are used for linking various conjugate groups to the nucleosides, oligonucleotides or oligonucleosides. Suitable conjugate groups include, but are not limited to, steroids, reporter molecules, reporter enzymes, lipophilic molecules, cleaver molecules, peptides and proteins.
Messenger RNA (mRNA) directs protein synthesis. Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
The naturally occurring events that provide the disruption of the nucleic acid function, discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989) are thought to be of two types. The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (Miller, et al., Anti-Cancer Drug Design 1987, 2, 117) and xcex1-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research reagents and therapeutic compounds. As research reagents oligonucleotides and oligonucleotide analogs find various uses including, but not limited to, probes and primers. For diagnostics, oligonucleotides and oligonucleotide analogs can be used in cell free systems, in vitro, ex vivo or in vivo. Currently a number of oligonucleotide based drugs are being tested in human clinical trials for various disease states including AIDS, against various cancers and for various systemic disease resulting from inappropriate immune responses. The antisense oligonucleotides and oligonucleotide analogs can be functionalized with various conjugate groups to modify certain of their properties. Thus reporter groups can be conjugated to the oligonucleotides or oligonucleotide analogs to assist in identification and location of the compounds in various testing medium including reagents, cellular products or digests, cell systems and organisms. Other conjugate groups can be utilized for transport, binding and uptake modulation, modification of solubility characteristics, analytical instrument identification and response and other useful properties known in the art.
Ramirez, et al., J. Am. Chem. Soc. 1982, 104, 5483, introduced the phospholipid group 5xe2x80x2-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3xe2x80x2 and 5xe2x80x2 positions. Subsequently Shea, et al., Nuc. Acids Res. 1990, 18, 3777, disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5xe2x80x2-phosphate on the 5xe2x80x2-terminus of the oligonucleotide. Certain of the Shea, et. al. authors also disclosed these and other compounds in patent application PCT/US90/01002. A further glucosyl phospholipid was disclosed by Guerra, et al., Tetrahedron Letters 1987, 28, 3581.
In other work, a cholesteryl group was attached to the inter-nucleotide linkage between the first and second nucleotides (from the 3xe2x80x2 terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and further by Letsinger, et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553. The aromatic intercalating agent anthraquinone was attached to the 2xe2x80x2 position of a sugar fragment of an oligonucleotide as reported by Yamana, et al., Bioconjugate Chem. 1990, 1, 319. The same researchers placed pyrene-1-methyl at the 2xe2x80x2 position of a sugar (Yamana et. al., Tetrahedron Lett. 1991, 32, 6347).
Lemairte, et al., Proc. Natl. Acad. Sci. USA 1986, 84, 648; and Leonetti, et al., Bioconjugate Chem. 1990, 1, 149. The 3xe2x80x2 terminus of the oligonucleotides each include a 3xe2x80x2-terminal ribose sugar moiety. The poly(L-lysine) was linked to the oligonucleotide via periodate oxidation of this terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates are described in European Patent application 87109348.0. In this instance the lysine residue was coupled to a 5xe2x80x2 or 3xe2x80x2 phosphate of the 5xe2x80x2 or 3xe2x80x2 terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3xe2x80x2 terminus of an oligonucleotide to link a peptide to the oligonucleotide as is described by Corey, et al., Science 1987, 238, 1401; Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; and Corey, et al., J. Am. Chem. Soc 1989, 111, 8524.
Nelson, et al., Nuc. Acids Res. 1989, 17, 7187 describe a linking reagent for attaching biotin to the 3xe2x80x2-terminus of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3xe2x80x2-Amine on. It is also commercially available under the name 3xe2x80x2-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991, 32, 879. A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5xe2x80x2-terminus of an oligonucleotide is 5xe2x80x2-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg, et al., Antisense Research and Development 1991, 1, 161 to link fluorescein to the 5xe2x80x2-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3xe2x80x2-terminus of an oligonucleotide. Asseline, et al., Proc. Natl. Acad. Sci. USA 1984, 81, 3297 described linking acridine on the 3xe2x80x2-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis, et al., Tetrahedron Letters 1987, 28, 5199 report building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3xe2x80x2 hydroxyl group of the 3xe2x80x2 terminal nucleotide of the oligonucleotide. Chollet, Nucleosides and Nucleotides 1990, 9, 957 attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the 5xe2x80x2 terminal phosphate of an oligonucleotide. They then used the bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukin protein to the oligonucleotide.
An EDTA iron complex has been linked to the 5 position of a pyrimidine nucleoside as reported by Dreyer, et al., Proc. Natl. Acad. Sci. USA 1985, 82, 968. Fluorescein has been linked to an oligonucleotide in the same manner as reported by Haralambidis, et al., Nucleic Acid Research 1987, 15, 4857 and biotin in the same manner as described in PCT application PCT/US/02198. Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser, et al., J. Am. Chem. Soc. 1989, 111, 6966. A commercial reagent, Amino-Modifier-dT, from Glen Research Corporation (Sterling, Va.) can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Carbamate linkages have been utilized to link conjugate groups to oligonucleotides at the 5xe2x80x2 position as reported by DeVos, et al., Nucleosides Nucleotides, 9, 259, 1990, Wachter, et al., Nucleic Acids Res., 14, 7985, 1986, and Gottikh, et. al., Tetrahedron Lett., 31, 6657, 1990. Carbamate linkages have not been used for link conjugate groups to the 2xe2x80x2 nor the 3xe2x80x2 position of nucleosides, however, carbamate linkages have been used to form fixed length 3xe2x80x2 to 5xe2x80x2 internucleoside linkage as reported by Stirchak et al., J. Orq. Chem., 52, 4202, 1987.
While currently utilized nucleosides, nucleotides and oligonucleotides conjugate linking moieties certainly have great utility, there is a continuing need for improved conjugate linking groups have a potpourri of different properties. One such property is as a transitory blocking group during oligonucleotide synthesis. A further property is to provide a foundation at the 2xe2x80x2 and 3xe2x80x2 positions where xe2x80x9cspacerxe2x80x9d molecules of various lengths, e.g., diaminoalkyl groups such as 1,2-ethylene diamine and 1,6-diaminohexane, can be attached for modifying the spacing between the nucleosides, nucleotides or oligonucleotides and the conjugate group.
It is one object of this invention to provide nucleosides, oligonucleotides and oligonucleosides that include carbamate chemical functionalities.
It is a further object of the invention to provide compounds for linking various conjugate groups via carbamate linkages to nucleosides, oligonucleotides and oligonucleosides.
It is another object to provide compounds that include conjugated intercalators, nucleic acid cleaving agents, cell surface phospholipids, and/or diagnostic agents.
It is yet another object to provide improvements in research and diagnostic methods and materials for assaying bodily states in animals, especially disease states.
It is an additional object of this invention to provide therapeutic and research materials having modified or improved spectral, solubility, transfer or uptake properties for the identification and analysis of DNA and RNA, for the diagnosis of normal or disease states of cells, cellular components or organisms and treatment of diseases through various mechanisms including modulation of the activity of DNA or RNA.
These and other objects are satisfied by the present invention, which provides compounds containing carbamate chemical functionalities. In one aspect, the invention provides oligonucleosides comprising a plurality of linked nucleosides, each of which includes a base portion and a ribofuranosyl sugar portion. In certain embodiments, at least one of such nucleosides bears at a 2xe2x80x2-O-position or a 3xe2x80x2-O-position a substituent having formula:
xe2x80x94RAxe2x80x94Nxe2x80x94C(X)xe2x80x94Oxe2x80x94R1a 
or 
xe2x80x94C(X)xe2x80x94N(R1b)(R1c) 
where:
RA is alkyl having from 1 to about 10 carbon atoms or (CH2xe2x80x94CH2xe2x80x94Q)x;
R1a is alkenyl having 2 to about 10 carbon atoms;
R1b and R1c, independently, are H, R2, RA, an amine protecting group or have formula RAxe2x80x94N(R1d)(R1e), C(X)xe2x80x94R2, C(X)xe2x80x94RAxe2x80x94R2, C(X)-Qxe2x80x94RAxe2x80x94R2, or C(X)-Qxe2x80x94R2;
R1d and R1a, independently, are H, R2, RA, an amine protecting group or have formula C(X)xe2x80x94R2, C(X)xe2x80x94RAxe2x80x94R2, C(X)-Qxe2x80x94RAxe2x80x94R2, or C(X)-Qxe2x80x94R2;
R2 is a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, includes folic acid or has formula -Q-(CH2CH2-Q-)x-R3;
X is O or S;
each Q is, independently, is NH, O, or S;
x is 1 to about 200;
R3 is H, RA, C(O)OH, C(O)ORA, C(O)R4, RAxe2x80x94N3, or RAxe2x80x94NH2;
R4 is Cl, Br, I, SO2R5 or has structure: 
m is 2 to 7; and
R5 alkyl having 1 to about 10 carbon atoms.
In other embodiments, at least one of the nucleosides of the compounds of the invention includes a pyrimidine base portion which bears at its 5-position a substituent having formula:
xe2x80x94RAxe2x80x94Oxe2x80x94C(X)xe2x80x94N(R1b)(R1c)
where RA, X, R1b, and R1c are as defined above.
The present invention also provides methods for inhibiting the expression of particular genes in the cells of an organism, comprising administering to said organism a compound according to the invention. Also provided are methods for inhibiting transcription and/or replication of particular genes or for inducing degradation of particular regions of double stranded DNA in cells of an organism by administering to said organism a compound of the invention. Further provided are methods for killing cells or virus by contacting said cells or virus with a compound of the invention. The compound can be included in a composition that further includes an inert carrier for the compound.
This invention provides nucleosides, oligonucleotides and oligonucleosides containing carbamate chemical functionalities. The nucleoside subunits can be xe2x80x9cnaturalxe2x80x9d or xe2x80x9csyntheticxe2x80x9d moieties. Each nucleoside is formed from a naturally occurring or synthetic base and a naturally occurring or synthetic pentofuranosyl sugar group.
The term xe2x80x9coligonucleotidexe2x80x9d refers to a polynucleotide formed from a plurality of linked nucleotide units. The nucleotides units each include a nucleoside unit. In the context of this invention, the term xe2x80x9coligonucleosidexe2x80x9d refers to a plurality of nucleoside units that are linked together. In a generic sense, since each nucleotide unit of an oligonucleotide includes a nucleoside therein, the term xe2x80x9coligonucleosidexe2x80x9d can be considered to be inclusive of oligonucleotides (i.e., nucleosides linked together via phosphate linking groups). In a further sense, the term xe2x80x9coligonucleosidexe2x80x9d also refers to a plurality of nucleosides that are linked together via linkages other than phosphate linkages. The term xe2x80x9coligonucleosidexe2x80x9d thus effectively includes naturally occurring species or synthetic species formed from naturally occurring subunits. For brevity, the term xe2x80x9coligonucleosidexe2x80x9d will be used as encompassing both phosphate linked (oligonucleotides) and non-phosphate linked polynucleoside species.
Oligonucleosides according to the invention also can include modified subunits. Representative modifications include modification of a heterocyclic base portion of a nucleoside or a sugar portion of a nucleoside. Exemplary modifications are disclosed in the following U.S. Pat. Nos. 5,138,045, 5,212,295, 5,223,618, 5,359,051, 5,359,044, 5,378,825, 5,457,191, 5,459,255, 5,489,677, 5,506,351, 5,519,134, 5,541,307, 5,543,507, 5,130,302, 5,134,066, 5,432,272, 5,457,187, 5,484,908, 5,502,177, 5,216,141, 5,434,257 and 3,687,808. The disclosure of each of these patents is incorporated herein by reference.
The term oligonucleoside thus refers to structures that include modified portions, be they modified sugar moieties or modified base moieties, that function similarly to natural bases and natural sugars. Representative modified bases include deaza or aza purines and pyrimidines used in place of natural purine and pyrimidine bases; pyrimidines having substituent groups at the 5- or 6-position; and purines having altered or replacement substituent groups at the 2-, 6-, or 8-positions. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at their 2xe2x80x2-position, and sugars having substituents in place of one or more hydrogen atoms of the sugar.
Altered base moieties or altered sugar moieties also include other modifications consistent with the spirit of this invention. Such oligonucleosides are best described as being structurally distinguishable from yet functionally interchangeable with naturally occurring or synthetic wild type oligonucleotides. All such oligonucleosides are comprehended by this invention so long as they function effectively to mimic the structure of a desired RNA or DNA strand.
The compounds of the present invention are those which bear a substituent having formula:
xe2x80x94RAxe2x80x94Nxe2x80x94C(X)xe2x80x94Oxe2x80x94R1a 
or 
xe2x80x94C(X)xe2x80x94N(R1b)(R1c) 
at a 2xe2x80x2-O- or 3xe2x80x2-O-nucleoside position or which bear a substituent having formula:
xe2x80x94RAxe2x80x94Oxe2x80x94C(X)xe2x80x94N(R1b)(R1c) 
at a 5-pyrimidine position.
RA can be alkyl having from 1 to about 10 carbon atoms or (CH2xe2x80x94CH2-Q)x, where Q is NH, O, or S and x is 1 to about 200, preferably 1 to about 50. Alkyl groups are substituents corresponding to branched and unbranched hydrocarbons. Preferred alkyl groups according to the invention have from 1, 2, or 6 carbon atoms.
R1a can be alkenyl having 2 to about 10 carbon atoms. Preferred alkenyl groups are those having 2 to about 5 carbon atoms. One particularly preferred alkenyl group is the 2-propenyl (i.e., xe2x80x94CH2CHxe2x95x90CH2) group.
R1b R1c, R1d, and R1e, independently, can be H, R2, RA, an amine protecting group or have formula RAxe2x80x94N(R1d)(R1e), C(X)xe2x80x94R2, C(X)xe2x80x94RAxe2x80x94R2, C(X)xe2x80x94Qxe2x80x94RAxe2x80x94R2, or C(X)xe2x80x94Qxe2x80x94R2. In preferred embodiments, R1b (and/or R1d) is H and R1c (and/or R1e) is H, R2, or RA, or R1b and R1c (and/or R1d and R1e), together, are a phthalimido amine protecting group. In other embodiments, R1d is H and R1e is C(X)xe2x80x94Qxe2x80x94R2 where X is S or, preferably, O, and Q is NH, O, or S.R1d is H and R1e is R2 or C(x)xe2x80x94Qxe2x80x94R2.
R2 is a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, or has formula xe2x80x94Qxe2x80x94(CH2CH2xe2x80x94Qxe2x80x94)x-R3. In preferred embodiments, R2 includes cholesterol or folic acid (i.e., includes a substantial portion of the cholesterol or folic acid molecule). For the purposes of this invention the terms xe2x80x9creporter moleculexe2x80x9d and xe2x80x9creporter enzymexe2x80x9d are inclusive of those molecules or enzymes that have physical or chemical properties that allow them to be identified in gels, fluids, whole cellular systems, broken cellular systems and the like utilizing physical properties such as spectroscopy, radioactivity, colorimetric assays, fluorescence, and specific binding. Steroids include those chemical compounds that contain a perhydro-1,2-cyclopentanophenanthrene ring system. Proteins and peptides are utilized in their usual sense as polymers of amino acids. Normally peptides comprise such polymers that contain a smaller number of amino acids per unit molecule than do the proteins. Lipophilic molecules include naturally-occurring and synthetic aromatic and non-aromatic moieties such as fatty acids, esters, alcohols and other lipid molecules, substituted aromatic groups such as dinitrophenyl groups, cage structures such as adamantane and buckminsterfullerenes, and aromatic hydrocarbons such as benzene, perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, and naphthacene.
Particularly useful as steroid molecules are the bile acids including cholic acid, deoxycholic acid and dehydrocholic acid; steroids including cortisone, digoxigenin, testosterone and cholesterol and even cationic steroids such as cortisone having a trimethylaminomethyl hydrazide group attached via a double bond at the 3-position of the cortisone rings. Particularly useful as reporter molecules are biotin, dinitrophenyl, and fluoresein dyes. Particularly useful as lipophilic molecules are steroid groups, alicyclic hydrocarbons, saturated and unsaturated fatty acids (such as palimitic and oleic), waxes, terpenes and polyalicyclic hydrocarbons including adamantane and buckminsterfullerenes. Particularly useful as reporter enzymes are alkaline phosphatase and horseradish peroxidase. Particularly useful as peptides and proteins are sequence-specific peptides and proteins including phosphodiesterase, peroxidase, phosphatase and nuclease proteins. Such peptides and proteins include SV40 peptide, RNaseA, RNase H and Staphylococcal nuclease. Particularly useful as terpenoids are vitamin A, retinoic acid, retinal and dehydroretinol.
Representative PEG groups are disclosed by Ouchi, et al., Drug Design and Discovery 1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and Delgardo et. al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249.
For use in antisense methodology, the oligonucleosides of the invention preferably comprise from about 10 to about 30 subunits. It is more preferred that such oligonucleosides comprise from about 15 to about 25 subunits. As will be appreciated, a subunit is a base and sugar combination suitably bound to adjacent subunits through, for example, a phosphorous-containing (e.g., phosphodiester) linkage or some other linking moiety. The nucleosides need not be linked in any particular manner, so long as they are covalently bound. Exemplary linkages are those between the 3xe2x80x2- and 5xe2x80x2-positions or 2xe2x80x2- and 5xe2x80x2-positions of adjacent nucleosides. Exemplary linking moieties are disclosed in the following references: Beaucage, et al., Tetrahedron 1992, 48, 2223 and references cited therein; and U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,034,506, 5,177,196, 5,214,134, 5,216,141, 5,264,423, 5,264,562, 5,264,564, 5,321,131, 5,399,676, 5,405,939, 5,434,257, 5,455,233, 5,476,925, 5,470,967, 5,495,009 and 5,519,126, as well as others of the above referenced patents. The disclosure of each of these patents is incorporated herein by reference.
It is preferred that the RNA or DNA portion which is to be modulated using oligonucleosides of the invention be preselected to comprise that portion of DNA or RNA which codes for the protein whose formation or activity is to be modulated. The targeting portion of the composition to be employed is, thus, selected to be complementary to the preselected portion of DNA or RNA, that is, to be an antisense oligonucleoside for that portion.
In accordance with one preferred embodiment of this invention, the compounds of the invention can be targeted to various mRNA sequences including those disclosed in U.S. Pat. Nos. 5,166,195, 5,242,906, 5,248,670, 5,442,049, 5,457,189, 5,510,239, 5,514,577, 5,514,788, 5,539,389 and 5,530,114. The disclosure of each of these patents is incorporated herein by reference.
The nucleosides and oligonucleosides of the invention can be used in diagnostics, therapeutics and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms, including warm-blooded animals, can be treated. Further, since each cell of multicellular eukaryotes can be treated since they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Many of the organelles (e.g., mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms. Thus, single cells, cellular populations or orgarelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides. As used herein, therapeutics is meant to include the eradication of a disease state, by killing an organism or by control of erratic or harmful cellular growth or expression.
In preparing compounds of the invention, one or more protecting groups can be used for temporary blocking a chemically reactive site in the molecules. Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as amine groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. See, e.g., Greene and Wuts, Protective Groups in Organic Synthesis, 2d edition, John Wiley and Sons, New York, 1991. Numerous amine protecting groups are known in the art, including, but not limited to: phthalimide (PHTH), trifluoroacetate (triflate), allyloxycarbonyl (Alloc), benzyloxycarbonyl (CBz), chlorobenzyloxycarbonyl, t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), and isonicotinyloxycarbonyl (i-Noc) groups. (see, e.g., Veber and Hirschmann, et al., J. Org. Chem. 1977, 42, 3286 and Atherton, et al., The Peptides, Gross and Meienhofer, Eds, Academic Press; New York, 1983; Vol. 9 pp. 1-38).
Oligonucleosides according to the invention can be assembled in solution or through solid-phase reactions, for example, on a suitable DNA synthesizer utilizing nucleosides according to the invention and/or standard nucleotide precursors. The nucleosides and nucleotide precursors can already bear alkylamino groups or can be later modified to bear such groups. Suitably protected nucleosides can be assembled into an oligonucleosides according to known techniques. See, e.g., Beaucage, et al., Tetrahedron 1992, 48, 2223.
Oligonucleosides according to the invention also can be prepared by assembling an oligonucleoside and appending an appropriate functionality thereto. For example, oligonucleosides having free hydroxyl groups can be assembled according to known techniques and then reacted with a reagent for linking the appropriate carbamate group thereto. As will be recognized, however, greater selectivity can be achieved in terms of placement of carbamate functionality within an oligonucleoside by introducing such functionality, as discussed above, on selected nucleosides and then using both the selected nucleosides and other nucleosides to construct an oligonucleoside.
Thus, the invention first builds the desired linked nucleoside sequence in the normal manner on the DNA synthesizer. One or more (preferably two or more) of the linked nucleosides are then functionalized or derivatized with the lipophilic steroid, reporter molecule, lipophilic molecule, reporter enzyme, peptide or protein.
Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. All oligonucleotide sequences are listed in a standard 5xe2x80x2 to 3xe2x80x2 order from left to right.